Beta arrestin‐related signalling axes are influenced by dexamethasone and metformin in vascular smooth muscle cells cultured in high glucose condition

Abstract Background Metformin (Met) and dexamethasone (Dexa) are known to reduce blood sugar levels and anti‐inflammatory effects, respectively. Based on the acceleration of atherosclerosis process in diabetes, the β‐arrestin 2 (BARR2) gene and protein expression levels were evaluated in vascular smooth muscle cells (VSMCs) treated with Met and Dexa in high glucose conditions in this study. Methods and Materials Human VSMCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM‐F12) medium and, were treated with different values of Met (1 mM, 5 mM and 7 mM) and Dexa (10−7 M, 10−6 M and 10−5 M) in 24‐ and 48‐h periods. The BARR2 gene and protein expression levels were identified with RT‐qPCR and western blotting techniques, respectively. The signalling axes were predicted from gene network made using Cytoscape software and, were annotated with Gene Ontology. Results The BARR2 gene and protein expression levels reduced in VSMCs treated with Dexa and Met after 24‐ and 48‐h periods. These results were more changed after 48 h. Furthermore, many BARR2‐related signalling axes were found from the network genes. Conclusion Met and Dexa suppressed the BARR2 protein and gene expression levels in the VSMCs. Moreover, the gene network suggested some the cellular signalling axes related to BARR2 that may be affected by Met and Dexa.


| INTRODUC TI ON
Atherosclerotic cardiovascular disease is known as one of the main causes of death in the world. 1 Some risk factors such as dyslipidaemia, hyperglycaemia, hypertension, genetics and, lifestyle reported to be involved in the development and progression of atherosclerosis process. 1,2From the point of molecular and cellular pathogenesis, atherosclerosis is known as a progressive event followed due to monocyte internalization through adhesion molecules, cellular polarization, formation and development of plaque within subendothelium space. 3Monocytes differentiate into macrophages and convert to foam cells due to scavenging the lipid particles such as Ox-LDL. 4The vascular smooth muscle cells (VSMCs) also play a key role in the development of atherosclerosis.These cells migrate into intima layer by stimulators such as interleukins, tumour necrosis factorα and platelet-derived growth factor. 5,68][9] It is also reported that β-arrestin 2 (BARR2), as a modulator of 7TM GPCRs, by activating MEK/ ERK signalling pathway in VSMCs promotes the atherosclerosis process. 102][13] Dexamethasone (Dexa) and other corticosteroids are also well recognized to affect immunological events 14 so that these drugs are suggested for the treatment of atherosclerosis. 15,16In addition, some studies showed that Dexa suppresses the migration and proliferation of VSMCs. 14,17,18K (PTK2), as a key gene in the chemokine signalling pathway, has recently reported to be involved in the proliferation and migration of VSMCs treated with Met. 19BARR2 is also a nuclear internalized protein in the chemokine signalling pathway so that we proposed Met and Dexa might affect its function in VSMCs.Based on the above descriptions, the aim of this study was to investigate the BARR2 gene and protein expression levels in VSMCs treated with Met and Dexa.In addition, the enriched gene networks were predicted to understand and explain the roles of BARR2 in the cellular signalling axes.
The cellular and molecular tests were repeated three times (n = 3).

| Gene networking
The work was followed on the results of FAK suppression in the Met-treated VSMCs. 19Similar to FAK (PTK2), BARR (ARRB), as an important central gene, was proposed to transduce the cellular signals from the upstream of chemokine signalling pathway (KEGG, hsa 04062).A primary gene network including the BARR and FAK were visualized by transferring the components of chemokine signalling pathway into Cytoscape (version 3.9.1)software using KEGGscape plugin.The network was enriched using CluGO plugin (Setting; Gene Ontology [GO], Molecular Function).Then, it was filtered in the CluGO plugin (GO hierarchy levels, 5-15) in other to limit the gene specificity on the GO-Molecular Function.The network genes were subjects for the prediction of involved pathways and, the molecular events in Reactome database (https:// react ome.org).

| RT-qPCR technique
Total RNA was extracted using GeneAll-Hybrid-R RNA purification kit (Cat.No. 305-101, Seoul, South Korea).Then, cDNA was synthesized using SMOBIO kit (Hsinchu, Taiwan).The BARR2 gene expression levels were measured by RT-qPCR technique (Applied Biosystems, Foster City, CA, USA) and, were normalized with GAPDH gene expression levels.The applied primers are shown in Table 1.

| Statistical analysis
Graphpad Prism (version 8.0.3) was used to analyse the study data.
Initially, the Kolmogorov-Simonov test was used to assess the data parametric distribution.Then, the data were compared using ANOVA.The BARR2 gene expression levels were calculated using 2-ΔΔCT formula.The data are presented in mean and standard deviation (mean ± SD).A p-value <.05 was estimated to be significant.

| Gene network found many cellular signalling axes related to β -arrestin 2
The BARR2 and FAK are contributed in the upstream of chemokine signalling pathway to transduce the cellular signals (Figure 1A).
The functions of network genes involved in the chemokine signalling pathway are shown in Figure 1B.The filtration of gene network showed that the FAK and ARRB act highly via nucleoside triphosphate-involved processes in the chemokine signalling pathway (Figure 1C).The Reactome data were annotated to the filtered genes so that there were many biological axes involved with the ARRB2 and FAK and their neighbour genes (Table 2).

| BARR2 gene expression levels reduced in VSMCs treated with metformin and dexamethasone
The BARR2 gene expression levels reduced significantly in the cell (VSMCs) groups treated with Met (1 mM, 5 mM and 7 mM) after 24 and 48 h as compared to the HG control group.However, more effects were observed in 48-h period.Also, the BARR2 gene expression levels reduced significantly in the cell (VSMCs) groups treated with Dexa (10 −5 M and 10 −6 M) after 48 h.Only the group treated with Dexa 10 −5 M reduced significantly the BARR2 gene expression levels after 24 h (p < .0001)(Figure 2).

| BARR2 protein expression levels reduced in VSMCs treated with metformin and dexamethasone
The BARR2 protein expression levels decreased in VSMCs treated with Met and Dexa.In the 24-h period, the BARR2 protein expression levels decreased significantly with values of Met 5 mM and 7 mM (p < .001),and Dexa 10 −5 M (p < .0001).Furthermore, BARR2 protein expression levels decreased significantly in the cell groups treated with Met (5 mM and 7 mM, p < .001)and Dexa (10 −5 M, 10 −6 M, 10 −7 M, p < .001) in the 48-h period (Figure 3; Figure S1).

| DISCUSS ION
1][22] Furthermore, some studies have shown that VSMCs involve in the stability and rupture of atherosclerotic plaques, and finally vessel stenosis. 23Some studies reported that the cellular growth factors and cytokines could affect the proliferation and migration of pathways in VSMCs. 5,24Moreover, hyperglycaemia is reported to be involved in the progression of atherosclerosis in diabetics.Other biological factors such as the activation of protein kinase C, the formation of advanced glycation end products (AGEs), and the stimulation of the lipoxygenase synthesis are suggested to relate to cardiovascular diseases. 7,25me studies reported that the HG-activated STAT3/Pim-1 signalling pathway mediates the VSMC proliferation and migration in HG conditions. 25Another study also reported that FAM3B stimulates the VSMC proliferation and migration in response to HG conditions. 26In contrast with the above studies, the VSMC proliferation and migration were not observed in HG conditions in a study, so that it proposed other subendothelial cells and modified compounds related to high blood sugar might be essential to improve the VSMC proliferation and migration. 27RR2, as a tumour suppressor, suppresses the mitogenic signalling pathway in prostate cancer cells.BARR2 also inhibits Src activation by CXCR7, resulting in the inhibition of EGFR-and ERKrelated signalling pathways. 28In addition, BARR2−/− mice revealed that VSMCs have lower proliferation than the wild type.These reports suggested that the inhibition of BARR2 might suppress the progression of atherosclerosis and vessel restenosis. 10t is widely used in type 2 diabetes and other disorders related to insulin resistance.Met affects oxidative stress, insulin sensitivity, gluconeogenesis, glucose absorption and consumption in the cells. 12,29Met may slow the progression of diabetes-related atherosclerosis by reducing VSMC proliferation and migration processes via mediating the AMPK pathway. 13There is also evidence that Met inhibits vascular calcification via AMPK/eNOS/NO axis. 11Dexa also has different biological effects, the most notable of which are the control and inhibition of immunological events.Several studies have demonstrated that Dexa can influence the function of VSMCs. 14,17,18ese reports found that raising the expression of PGC-1 18 and modifying the activity of matrix metalloproteinase inhibit the proliferation and migration of VSMCs. 17The results of this study showed that Met and Dexa suppress the BARR2 protein and gene expression levels.Since BARR2 is known to involve in cell growth, 28 thus the study suggested that Met and Dexa might inhibit the VSMC proliferation and migration.The study also suggested the cellular events related to BARR2 might be involved in many signalling axes.

| CON CLUS ION
The results showed that Met similar to Dexa suppresses the BARR2 protein and gene expression levels in the VSMCs.Moreover, the gene network showed that the BARR2 is a central gene to transduce the signalling messages in the chemokine signalling pathway so that the decrease of BARR2 expression level suppresses the chemokine signalling pathway via the nucleoside triphosphate metabolism as shown in the enrichment of gene network.Furthermore, the gene network showed that the BARR2-related signalling axes might affect many cellular signalling pathways.The prediction results suggested that the decrease of BARR2 by Met and Dexa might affect the chemokine signalling pathway and other BARR2-related signalling axes.However, more studies are needed to clear the roles of Met and Dexa in these predicted pathways.
Total protein was extracted by RIPA buffer (Cat.No. sc-24,948, Santa Cruz Biotechnology, USA) from the cell groups.Then, the protein samples together with prestained Protein Ladder (SL7001, SinaClon, Iran) were run on SDS-PAGE with 8-well comb (#1704463, BIO-RAD) and, were transferred on PVDF membrane (IPVH00010, Merck Millipore, Darmstadt, Germany).The primary (#3857, 1:1000, Cell Signalling Technology) and conjugated secondary antibodies (#7074, 1:10000, Cell Signalling Technology) were used to detect BARR2.The protein blot was visualized with the ECL reagents (RPN2235, AmershamGeneForward primer Reverse primer GAPDH CATGA GAA GTA TGA CAA CAGCCT AGTCC TTC CAC GAT ACC AAAGT BARR2 GTCAA GGT GAA GCT GGTGGT ATGAG GTT GGT GTC CACAGG TA B L E 1 Primer sequences.F I G U R E 1 Gene networking.(A) The ARRB (BARR) and PTK2 (FAK) genes in the upstream of chemokine signalling pathway.(B) The enrichment of genes in chemokine signalling pathway using molecular function (GO, Levels 5-15).(C) The filtration of chemokine signalling pathway based on the presence of ARRB and PTK2 genes in the enriched gene network.Biosciences, Italy) and, was quantified by ImageJ software.Data were normalized with beta-actin (#4967, Cell Signalling Technology).

F I G U R E 2
β-arrestin 2 (BARR2) gene expression levels in vascular smooth muscle cells (VSMCs) treated with dexamethasone (Dexa) and metformin (Met) for 24 h and 48 h.(A) After 24 h of treatment.The BARR2 gene expression levels reduced significantly in the cell groups treated with Met (5 and 7 mM) and Dexa (10 −5 M). (B) After 48 h.The BARR2 gene expression levels reduced in the cell groups treated with Met (5 and 7 mM) and Dexa (10−5 and 10−6 M).The tests are repeated three times (n = 3).The data are presented in Mean and Standard deviation (mean ± SD). *p < .05;**p < .001;***p < .0001.HG, high glucose.F I G U R E 3 β-arrestin 2 (BARR2) protein expression levels in vascular smooth muscle cells (VSMCs) treated with dexamethasone (Dexa) and metformin (Met) for 24 h and 48 h.(A) Western blot image.(B) After 24 h of treatment.The BARR2 protein expression levels reduced significantly in the VSMC groups treated with Met (5 and 7 mM) and Dexa (10 −5 M). (C) After 48 h.The BARR2 protein expression levels reduced in the all of cell groups treated with Dexa (10 −5 and 10 −6 and 10 −7 M) and Met (5 and 7 mM).The tests are repeated three times (n = 3).The data are presented in Mean and Standard deviation (mean ± SD). *p < .05;**p < .001;***p < .0001.HG, high glucose.